The delivery of genetic material as a therapeutic promises to be a revolutionary advance in the treatment of disease. Although, the initial motivation for gene therapy was the treatment of genetic disorders, it is becoming increasingly apparent that gene therapy will also be useful for the treatment of a broad range of acquired diseases such as cancer, infectious disorders (AIDS), heart disease, arthritis, and neurodegenerative disorders (Parkinson's and Alzheimer's). In addition to providing a therapeutic benefit, nucleic acid delivery is also beneficial for the study of gene function and drug interaction. Not only can functional genes be delivered to repair a genetic deficiency, but polynucleotides can also be delivered to inhibit gene expression. Inhibition of gene expression can be affected by antisense polynucleotides, siRNA mediated RNA interference and ribozymes. Transfer methods currently being explored included viral vectors and physical-chemical methods.
RNA interference (RNAi) describes the phenomenon whereby the presence of double-stranded RNA (dsRNA) of sequence that is identical or highly similar to a target gene results in the degradation of messenger RNA (mRNA) transcribed from that target gene [Sharp 2001]. It has been found that RNAi in mammalian cells is mediated by short interfering RNAs (siRNAs) of approximately 21-25 nucleotides in length or microRNAs [Tuschl et al. 1999; Elbashir et al. 2001]. The ability to specifically inhibit expression of a target gene by RNAi has obvious benefits. For example, RNAi could be used to study gene function. In addition, RNAi could be used to inhibit the expression of deleterious genes and therefore alleviate symptoms of or cure disease. SiRNA/microRNA delivery may also aid in drug discovery and target validation in pharmaceutical research.
A basic challenge in gene therapy, whether for therapeutic or research purposes, is to develop approaches for delivering genetic material to the appropriate cells in a way that is specific, efficient and safe. This problem of ‘drug delivery’, where the drug is a polynucleotide, is particularly challenging. If polynucleotides are appropriately delivered they can potentially lead to amelioration of a disease or condition. Delivery of polynucleotides to cells in vivo is also useful in basic research: in the study of cell physiology, gene function, drug discovery and interaction, disease, and infection.
Gene therapy promises to be a significant advance in the treatment of both acquired and genetic diseases at the most fundamental levels of pathology. Specifically, the development of gene transfer methods into the heart is attractive given that coronary artery disease is the leading cause of morbidity and mortality in the United States. Despite advances in the prevention and treatment of this disorder there remains a large population of patients who are not optimal candidates for percutaneous or surgical revascularization, usually because of severe distal vessel disease or previous failed revascularization procedures. Coronary collateral development is an important adaptive response of the ischemic heart in this situation, but often the collateral circulation is inadequate and results in severe angina pectoris despite maximal medical therapy. A new strategy to treat these often disabled patients involves the local delivery of vascular cytokines to induce new blood vessel growth (neoangiogenesis) in the ischemic myocardium. Alternatively, nucleic acids (e.g., siRNA or siRNA expression vectors) can be delivered that down regulated repressors of angiogenesis. It has been recognized that gene therapy could play a major role in neovascularization approaches.
A variety of techniques has been developed to transfer polynucleotides into the heart. These techniques have principally involved adenovirus vectors, which can be injected directly or intravenously, and plasmid DNA vectors injected directly into the heart tissue. The first reports of successful non-viral in vivo gene delivery to the heart used direct injection of plasmid DNA vectors [Lin et al. 1990; Acsadi et al. 1991]. High levels of β-galactosidase reporter gene expression were measured several days after injection of plasmid DNA solutions. Expression appeared to be highly localized to the site of injection. Adenoviral vectors have been used extensively for gene transfer into cardiac muscle. Barr et al. found transduction levels of 10-32% after intracoronary installation of virus. However, expression was also found in endothelial cells and the presence of the viral genome was detected in other organs [Barr et al. 1994]. Many of these gene therapy studies were aimed at transducing vascular endothelial cells to prevent restenosis following angioplasty. The injection of adenoviral vectors into the portal or systemic circulatory systems leads to high, but transient levels of foreign gene expression in several organs (liver, lung, etc.). Immune responses directed against the viral coat proteins and proteins expressed from the viral genome are another problem associated with viral delivery. Also, adenoviral transduction of infarcted heart tissue has been less efficient than normal tissue. Decreased transduction efficiency in diseased heart tissue could be a problem for viral gene therapy approaches for ischemic heart disease.
Until recently, the direct injection of plasmid DNA into the heart has mainly been used to benefit basic researchers investigating transcriptional regulation of cardiac specific genes. Isner and co-workers [Takeshita S et al. 1994] have pioneered the in vivo delivery of genes that result in neovascularization of ischemic muscle. In a breakthrough gene therapy study, they demonstrated significant formation of new vessels, enhanced distal flow, and clinical benefit in patients with ischemic limbs following injection of plasmid DNA expressing the human vascular endothelial growth factor (hVEGF) gene [Baumgartner I et al. 1998]. This same hVEGF-expressing plasmid has been injected into ischemic heart tissue in humans [Losordo DW et al. 1998]. Preliminary results are very promising, with significant reduction in reported angina, and improved Rentrop score in 5 of 5 patients.
In vivo transfection of plasmid DNA complexed with liposomes after direct injection into heart muscle resulted in localized expression of reporter genes [Aoki M et al. 1997]. While highly effective in vitro, liposome-complexed plasmid DNA particles generally have been of limited success in vivo. Systemic delivery of both adenoviral vectors and liposome-plasmid DNA complexes provides much greater efficiency of delivery into liver and lung than into heart, making these strategies unattractive for cardiac gene therapy.
Using a porcine chronic coronary occlusion model, it was shown that single coronary injections of a replication-defective adenovirus vector expressing fibroblast growth factor-5 (FGF-5) resulted in improved regional myocardial blood flow and histological evidence of increased capillary number [Giordano F J et al. 1996]. Stress-induced regional contractile dysfunction was documented by echocardiography after coronary occlusions in this study. Importantly, this pacing-induced regional wall motion abnormality was completely normalized within two weeks after intracoronary FGF-5 gene transfer and the amelioration of stress-induced myocardial ischemia persisted for at least 12 weeks. Again using the porcine model chronic coronary occlusion model, Mack et al. 1998 showed similar amelioration of pacing-induced myocardial ischemia and improvement in blood flow after multiple direct intramyocardial injections of a replication-defective adenovirus expressing hVEGF121.
The intravascular delivery of plasmid DNA has been shown to be highly effective for gene transfer into liver, skeletal and cardiac muscle (U.S. application Ser. No. 09/330,909, U.S. Pat. No. 6,627,616). Non-viral vectors are inherently safer than viral vectors, have a reduced immune response induction and have significantly lower cost of production. Furthermore, a much lower risk of transforming activity is associated with non-viral polynucleotides than with viruses.